We are attempting to develop a solid phase ELISA system for the detection and quantitation of immunoglobulins in human stool specimens directed against rotavirus. Major efforts to date have been directed at establishing reliable and optimal procedures and reagents for each step in this multi-layered """"""""sandwich"""""""" assay. The basic test system is: 1) precoat 96 well plates with hyperimmune goat 930 rotavirus antiserum as capture antibody, 2) add rotavirus antigen, 3) add test specimens of serum or stool containing antibodies to rotavirus, 4) add antibodies to human immunoglobulin conjugated to peroxidase and 5) add specific substrate for peroxidase which causes development of color that can be read as optical density units. To date several difficulties have been encountered. The most confounding difficulty has been the long unrecognized intermittent partial/total failure of the conjugate. One other problem is that of high background color due to non-specific interactions with the precoat and several modifications will be explored to overcome this difficulty including use of purified rotavirus antigen, treatment to block """"""""nonspecific"""""""" sites, change in the order of the ELISA sandwich layers.
